Our long-term goal is to understand mechanisms directing the initiation and morphogenesis of the extra-ocular muscles and guiding appropriate motor axons to them, defects of which contribute to congenital oculomotor diseases such as Duane syndrome. Our hypothesis is that a consortium of positive and negative myogenic signals including highly localized signals from the brain (e.g., fgf8) act upon adjacent paraxial mesoderm cells. Our preliminary data show that altering signaling properties of the avian midbrain by ectopic expression of fgf8 disrupts development of the dorsal oblique (DO). We will screen for putative myogenic signals in the avian embryo, then test candidates by introducing signal agonists or antagonists into the midbrain (for DO induction) or hindbrain (for lateral rectus, LR, induction). This will be done using electroporation and infection with RCAS retroviral constructs. To examine the response capabilities of muscle primordia, trunk mesoderm infected with response blocking constructs, e.g. sprouty1, will be transplanted into infection-resistant strains of chick embryos in place of the normal LR primordium. Embryos are assayed for several markers of extra-ocular muscle differentiation, for changes in gene expression and neurogenesis in the brain, and for patterns of axon outgrowth. Cranial nerves In, IV, and VI arise in separate locations and each shows patterns of early outgrowth that are unique. Our hypothesis is that outgrowing abducens and oculomotor axons use different combinations of guidance systems, including growth factor-receptor pairs and members of the slit-robo, semaphorin-neuropilin, and ephrin-eph families. After screening for candidate guidance molecules, we will test for prespecification of motor axon properties by transplanting motor neuron precursors between sites of nerve III, VI, and XIII formation, then evaluate the roles of identified guidance molecules by creating changes in candidate gene expression in motor neurons using electroporation. Aim 3 exploits an avian model of the cross-innervation of the LR muscle by oculomotor axons that accompanies Duane syndrome. This is produced by a simple early lesion to the abducens primordium. Our hypothesis is that in the absence of the abducens nerve, there is an upregulation of neurorepellants and/or downregulation of neuroattractants within the LR that allows n. III ingrowth. We will screen for candidate molecules, especially HGF and GDNF, then test for their roles by overexpressing neurotrophic candidates into the LR primordium.